Recombinant Chimeric Chicken Antibodies

The chicken IgY Fc region has unique and advantageous characteristics.

The constant (Fc) regions are responsible for effector functions such as receptor binding, complement activation, and interaction with rheumatoid factors. There are significant functional differences between the Fc regions of mammalian IgG and chicken IgY:

1. Secondary antibodies raised against mammalian IgG will not cross-react with chicken IgY.
2. Unlike mammalian IgG Fc, chicken IgY Fc does not bind to mammalian Fc receptors, interact with rheumatoid factors, or activate the mammalian complement system.

These differences in effector function offer practical advantages in many experimental applications, such as the ease of using mouse-derived antigen-binding sequences on mouse tissues, easier multiplexing, and reduced background.

Chimeric antibodies: production and therapeutics

A chimeric antibody is created by using recombinant DNA technologies to generate expression plasmids encoding the desired regions of each cloned parental antibody.

Much of the initial work in the area of chimeric antibodies was driven by a desire to produce therapeutic antibodies with more human properties. In the first published report of a chimeric antibody, mouse antigen-binding domains were combined with human constant region domains (Morrison , S.L. et al (1984) PNAS 81, 6851-6855). This generates mouse-human chimeras with reduced immunogenicity when injected into humans. Although the majority of marketed therapeutic antibodies are now more fully humanized, a number of mouse-human chimeras have been approved for therapeutic use in humans. This can be seen as a powerful validation of chimeric antibody technology.

The use of chimeric antibody technology is not limited to therapeutic applications, it also enables the creation of research-use antibodies with the unique and favorable properties mentioned above.

Chimeric chicken antibodies as research tools

Although chimeric antibodies were first described in the 1980s, mammalian-chicken chimeric antibodies as commercially available research tools are a recent development, following on the heels of the introduction of non-chimeric recombinant antibodies.

Given the evolutionary distance between mammalian and avian immunoglobulins it is not unreasonable to ask whether combining mouse variable (antigen-binding) regions with chicken constant regions has any adverse effect of the antigen-binding properties of the chimeric antibody. This question was addressed by Choi et al (2019 Scientific Reports 9, 19242). Taking two mouse monoclonal antibodies, the authors created mouse-chicken and mouse-human chimeric versions. The paper demonstrated that any changes in antigen binding affinities (measured by surface plasmon resonance) compared to the parental mouse antibodies were small and remarkably similar for both the mouse-chicken chimeras and the mouse-human chimeras. This is a quantitative demonstration of the interchangeability of avian and mammalian constant regions.

Advantages of our chimeric chicken antibodies

Our chimeric chicken antibodies offer all the advantages associated with recombinant antibodies while also giving you more flexibility for multiplex imaging with lower background.

1. Reproducibility: Because our chicken chimeric antiboides are recombinant, they offer a consistent, reproducible, inexhaustible source of antibody. Exactly the same antibody sequence will be produced time-after-time, batch-after-batch ensuring you consistently obtain reproducibile data.
2. Animal-friendly: Our chimeric chicken antibodies are produced in vitro using serum-free and protein-free growth media. This means that no animals are used in their production, making them a humane and ethical source of antibodies.
3. Known binding properties: The antibody variable regions - which contain the antigen-binding sites - are from rigorously validated mouse monoclonal antibodies, so the antigen binding properties are known to generate reliable, high-quality data.
4. Reduced background/enhanced performance: There are distinct functional properties of the chicken IgY Fc region that provide an advantage over antibodies containing a mammalian IgG Fc region.
   • IgY Fc does not bind to mammalian Fc receptors. This avoids antigen-independent binding of mammailian immune cells expressing FcγR receptors and reduces background.
   • IgY Fc does not interact with rheumatoid factors. Not binding to these auto-antibodies recognizing the Fc region of mammalian IgG means fewer false positives in animal serum samples.
   • IgY Fc does not activate the mammalian complement system. This reduces signal interference in ELISA assays and also can be advantageous in in-vivo applications.
     (See Larsson et al (1993) (Poultry Science 72 (10), 1807-1812; Pereira et al (2019) International Immunopharmacology 73, 293-303).
5. Facilitates staining of mouse tissues: Using mouse antibodies to stain mouse samples presents challenges since anti-mouse secondary antibodies can react with endogenous mouse IgG in the sample resulting in background (the so called “mouse-on-mouse” issue). While methods exist for reducing such background, our chimeric chicken antibodies avoid this issue entirely since anti-chicken IgY secondary antibodies will not bind to endogenous mouse IgG.
6. Better multiplexing with chicken antibodies: Simultaneously staining for multiple targets in a sample (multiplexing) requires that each anti-target antibody be differentially labeled to allow each target in the sample to be individually resolvable. This can be accomplished by directly conjugating a reporter (fluorescent dye, enzyme etc…) to each individual anti-target antibody or through the use of indirect immunfluoresence using fluorescently-labeled secondary antibodies. The latter approach is often used since multiple fluorescently-labeled secondary antibodies can bind to a single ‘primary’ antibody resulting in signal amplification and a stronger/brighter signal. Since most commercial antibodies are from mouse or rabbit this generally limits indirect fluorescent multiplexing to two targets, one recognized by an anti-mouse secondary antibody and one recognized by an anti-rabbit secondary antibody. Chicken antibodies expand the size of the multiplex panel that can be used for indirect immunofluorescence and allow you to get more data from your samples with less background!  

A note about immunoprecipitation (IP) and Chimeric Chicken Antibodies

One potential disadvantage to chimeric chicken antibodies is the fact that the Fc region of chicken IgY does not bind to Protein A or Protein G, reagents commonly used in immunoprecipitation protocols with mammalian IgG antibodies. However, this can be readily overcome by using our PrecipHen® Immunoprecipitation Reagent (made from goat anti-chicken IgY antibodies covalently attached to agarose beads) and designed for use with chicken antibodies in immunoprecipitation (IP), chromatin immunoprecipitation (ChIP), and protein purification applications.